First baseline data of the Klinefelter ItaliaN Group (KING) cohort: clinical features of adult with Klinefelter syndrome in Italy.

BACKGROUND: Klinefelter syndrome (KS) is frustratingly under-diagnosed. KS have a broad spectrum of clinical features, making it difficult to identify.  OBJECTIVE: We describe KS clinical presentation in a large Italian cohort. DESIGN: This is the first observational cohort study within a national network, the Klinefelter ItaliaN Group (KING). Primary outcomes were to describe the basic clinical features and the actual phenotype of KS in Italy. Secondary outcomes were to determine age at diagnosis and geographical distribution. METHODS: We performed a basic phenotyping and evaluation of the hormonal values of 609 adult KS patients. RESULTS: Mean age at diagnosis was 37.4 ± 13.4 years. The overall mean testicular size was 3 ml, and 2.5 ml in both testes in untreated KS group. BMI was 26.6 ± 5.8 kg/m2, and 25.5% of KS had metabolic syndrome (MetS). LH and FSH were increased, and mean total testosterone were 350 ± 9.1 ng/dl. A descriptive analysis showed that 329 KS patients were evaluated in Northern Italy, 76 in Central and 204 in Southern Italy. Analysis of variance demonstrated significant statistical differences (p < 0001) between the age at diagnosis of the three geographical groups. Compared with the expected number among male patients matched for age in Italy, only 16% of KS patients received a diagnosis. CONCLUSIONS: These data are the results of the only national database available that collects the clinical and hormonal data of the KS patients, currently referred at the KING centers. In Italy the typical KS patient is overweight, with small testes, and elevated LH and FSH. Only 25.5% of them are diagnosed with MetS. Early detection and timely treatment are mandatory.

Risk factors on testicular function in adolescents.

PURPOSE: Adolescence represents an important window for gonadal development. The aim of this review is to carry out a critical excursus of the most recent literature on endogenous and exogenous risk factors related to testicular function, focusing the research on adolescence period. METHODS: A comprehensive literature search within PubMed was performed to provide a summary of currently available evidence regarding the impact on adolescence of varicocele, cryptorchidism, cancer, diabetes, lifestyle factors, endocrine disruptors, obesity and sexually transmitted diseases. We focused on human studies that evaluated a possible impact of these factors on puberty timing and their effects on andrological health. RESULTS: Evidence collected seems to suggest that andrological health in adolescence may be impaired by several factors, as varicocele, cryptorchidism, and childhood cancer. Despite an early diagnosis and treatment, many adolescents might still have symptoms and sign of a testicular dysfunction in their adult life and at the current time it is not possible to predict which of them will experience andrological problems. Lifestyle factors might have a role in these discrepancies. Most studies point out towards a correlation between obesity, insulin resistance, alcohol, smoking, use of illegal drugs and testicular function in pubertal boys. Also, endocrine disruptors and sexually transmitted diseases might contribute to impair reproductive health, but more studies in adolescents are needed. CONCLUSION: According to currently available evidence, there is an emerging global adverse trend of high-risk and unhealthy behaviors in male adolescents. A significant proportion of young men with unsuspected and undiagnosed andrological disorders engage in behaviors that could impair testicular development and function, with an increased risk for later male infertility and/or hypogonadism during the adult life. Therefore, adolescence should be considered a key time for intervention and prevention of later andrological diseases.

Perfluorooctanoic acid alters progesterone activity in human endometrial cells and induces reproductive alterations in young women.

Female fecundity is finely regulated by hormonal signaling, representing a potential target for endocrine-disrupting chemicals. Among the chemicals of most concern are the perfluoroalkyl substances (PFAS), widely used in consumer goods, that are associated with adverse effects on reproductive health. In this context, the endometrium clearly represents an important fertility determining factor. The aim of this study was to investigate PFAS interference on hormonal endometrial regulation. This study was performed within a screening protocol to evaluate reproductive health in high schools. We studied a cohort of 146 exposed females aged 18-21 from the Veneto region in Italy, one of the four areas worldwide heavily polluted with PFAS, and 1080 non-exposed controls. In experiments on Ishikawa cells included UV-Vis spectroscopy, microarray analysis and qPCR. We report a significant dysregulation of the genetic cascade leading to embryo implantation and endometrial receptivity. The most differentially-expressed genes upon PFOA coincubation were ITGB8, KLF5, WNT11, SULT1E1, ALPPL2 and G0S2 (all p < 0.01). By qPCR, we confirmed an antagonistic effect of PFOA on all these genes, which was reversed at higher progesterone levels. Molecular interference of PFOA on progesterone was confirmed by an increase in the intensity of absorption spectra at 250 nm in a dose-dependent manner, but not in the presence of ß-estradiol. Age at menarche (+164 days, p = 0.006) and the frequency of girls with irregular periods (29.5% vs 21.5%, p = 0.022) were significantly higher in the exposed group. Our results are indicative of endocrine-disrupting activity of PFAS on progesterone-mediated endometrial function.

Perfluoro-octanoic acid impairs sperm motility through the alteration of plasma membrane.

CONTEXT: Perfluoroalkyl-substances (PFAS) are chemical additives considered harmful for humans. We recently showed that accumulation of perfluoro-octanoic acid (PFOA) in human semen of exposed subjects was associated with altered motility parameters of sperm cells, suggesting direct toxicity. OBJECTIVES: To determine whether direct exposure of human spermatozoa to PFOA was associated to impairment of cell function. PATIENTS AND METHODS: Spermatozoa isolated from semen samples of ten normozoospermic healthy donors were exposed up to 2 h to PFOA, at concentrations from 0.1 to 10 ng/mL. Viability and motility parameters were evaluated by Sperm Class Analyser. Cell respiratory function was assessed by both mitochondrial probe JC-1 and respiratory control ratio (RCR) determination. Sperm accumulation of PFOA was quantified by liquid chromatography-mass spectrometry. Expression of organic ion-transporters OATP1 and SLCO1B2 was assessed by immunofluorescence and respective role in PFOA accumulation was evaluated by either blockade with probenecid or membrane scavenging through ß-cyclodextrin (ß-CD). Plasma membrane fluidity and electrochemical potential (ΔΨp) were evaluated, respectively, with Merocyanine-540 and Di-3-ANEPPDHQ fluorescent probes. RESULTS: Compared to untreated controls, a threefold increase of the percentage of non-motile sperms was observed after 2 h of exposure to PFOA regardless of the concentration of PFOA, whilst RCR was significantly reduced. Only scavenging with ß-CD was effective in reducing PFOA accumulation, suggesting membrane involvement. Altered membrane fluidity, reduced ΔΨp and sperm motility loss associated with exposure to PFOA were reverted by ß-CD treatment. CONCLUSION: PFOA alters human sperm motility through plasma-membrane disruption, an effect recovered by incubation with ß-CD.

Risk behaviours and alcohol in adolescence are negatively associated with testicular volume: results from the Amico-Andrologo survey.

BACKGROUND: Risk factors established during adolescence affect health outcomes in adulthood, although little is known about how adolescent health risk behaviours (HRBs) affect testicular development and reproductive health. OBJECTIVES: To assess prevalence of HRBs among last year high school students; to describe the most prevalent andrological disorders in this cohort; to explore HRBs associated with andrological disorders and investigate factors possibly associated with impaired testicular development in puberty. MATERIALS AND METHODS: The Amico-Andrologo Survey is a permanent nationwide surveillance programme conducted by the Italian Society of Andrology and Sexual Medicine and supported by the Ministry of Health. A nationally representative survey of final-year male high school students was conducted using a validated structured interview (n = 10124) and medical examination (n = 3816). RESULTS: Smoking (32.6%), drinking (80.6%) and use of illegal drugs (46.5%) are common in adolescence. 16.6% of subjects were overweight, 3.1% were underweight and 2.3% were obese. Among sexually active students (60.3%), unprotected sex was very common (48.3%). Only 11.6% had been treated for andrological disorders, despite an abnormal clinical examination in 34.6%. Bilateral testicular hypotrophy (14.0%), varicocoele (27.1%) and phimosis (7.1%) were the most prevalent disorders; 5.1% complained of premature ejaculation and 4.7% had an STI. Underweight and heavy alcohol or drug use were associated with testicular hypotrophy. HRBs emerged as significant predictors of testicular hypotrophy, explaining up to 9.6% of its variance. Limitations include risk of selection bias for voluntary physical examination and recall bias for the self-compiled questionnaire. DISCUSSION: There is an emerging global adverse trend of HRBs in male high school students. A significant proportion of adolescent males with unsuspected andrological disorders engage in behaviours that could impair testicular development. CONCLUSION: Greater attention to the prevention of andrological health in adolescence is needed.

E2F1 copy number variations contribute to spermatogenic impairment and cryptorchidism by increasing susceptibility to heat stress.

BACKGROUND: Copy number variations (CNVs) play an important role in the onset of several diseases, and recently research focused on the relationship between these structural variants and diseases of the reproductive tract, including male infertility and cryptorchidism. OBJECTIVES: To evaluate the contribution of copy number variations of E2F1 gene to idiopathic male infertility and the factors influencing expression of this gene. MATERIALS AND METHODS: We performed a retrospective study on 540 subjects recruited from September 2014 to February 2015. TaqMan CNV assay was used to analyze E2F1 CNV. Real-time PCR was used to assess E2F1 and HSP70 expression level in heat stressed and transfected cells with three E2F1 copies. RESULTS: We found a significant difference in the frequency of altered E2F1 copies in patients (12/343, 3.5%) compared with controls (0/197) (p = 0.005). Six patients with E2F1 CNV had history of cryptorchidism, but the prevalence between men with idiopathic infertility (6/243, 2.5%) and infertile men with history of cryptorchidism (6/100, 6.0%) was not statistically different (p = 0.1). E2F1 expression increased under heat stress conditions, especially in cells carrying more copies of gene and this was associated with increased expression of HSP70. DISCUSSION: Our data suggest that an abnormal E2F1 expression caused by multiple copies of E2F1 gene predisposes to the onset of infertility and that the risk further increases if subjects with altered E2F1 copies have stressful conditions, such as heat stress or history of cryptorchidism. CONCLUSION: This study shows a link between E2F1 CNV and male infertility, suggesting that the increased risk of spermatogenic impairment associated with higher E2F1 copies might be due to higher susceptibility to stressful conditions.

Anogenital distance is associated with genital measures and seminal parameters but not anthropometrics in a large cohort of young adult men.

STUDY QUESTION: Is the anogenital distance (AGD) correlated to anthropometric, genital and sperm parameters in young adult men? SUMMARY ANSWER: We observed that reduced AGD is strongly associated with altered semen parameters and reduced testicular volume. WHAT IS KNOWN ALREADY: Abnormalities in the foetal development of the testis have been suggested as causative of common male reproductive disorders, such as cryptorchidism, hypospadias, reduced semen quality and testicular germ cell tumour, collectively defined as 'testicular dysgenesis syndrome'. In human epidemiological studies, alterations in AGD have been frequently associated with clinically relevant outcomes of reproductive health, suggesting AGD as a marker of foetal testicular development. STUDY DESIGN, SIZE, DURATION: This study was performed within the annual screening protocol to evaluate male reproductive health in the high schools of Padua and surroundings (Veneto Region, the North-East of Italy). Here we report the findings of 794 subjects who completed the study protocol between October 2016 and May 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated 794 students aged 18-19 years recording the following parameters: height, weight, BMI, waist circumference, arm span, pubis-to-floor and crown-to-pubis length, penile length and circumference, testicular volumes, semen parameters and AGD (measured from the posterior base of the scrotum to the centre of the anus). MAIN RESULTS AND THE ROLE OF CHANCE: Of the subjects, 49% had an abnormal arm span-height difference (>3 cm) and 63.4% had an altered ratio of crown-to-pubis/pubis-to-floor length (≤0.92). The rate of subjects with reduced testicular volume was 23%. Median sperm concentration was 51.0× 106/ml and total sperm count was 122.5 × 106. AGD showed a direct positive relation with testicular volume and penile length and circumference (R = 0.265, 0.176 and 0.095, respectively, all P < 0.05). No significant relation was observed between AGD and anthropometric parameters. Sperm concentration, total sperm count, progressive motility and normal morphology showed a significant and positive correlation with AGD (R = 0.205, 0.210, 0.216 and 0.117, respectively, all P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Our cohort of young adults is not representative of the general population. Hormonal evaluation was missing. WIDER IMPLICATIONS OF THE FINDINGS: Our findings show that AGD is associated with testicular volumes, penile measures and seminal parameters in young adult men. Because AGD is hormonally determined during foetal life, the reported high incidence of reduced semen quality and reduced testicular volume could be related to a reduced androgenic exposure in utero. AGD could represent a simple and useful method to evaluate testicular and penile development in adult men. STUDY FUNDING/COMPETING INTEREST(S): The authors have no potential conflict of interest to declare. No external funding was obtained for this study. TRIAL REGISTRATION NUMBER: N/A.

Hypovitaminosis D is associated with lower urinary tract symptoms and benign prostate hyperplasia in type 2 diabetes.

Lower urinary tract symptoms (LUTS) may develop more commonly in men with type 2 diabetes mellitus (T2DM). LUTS are often associated with benign prostate hyperplasia (BPH), in general population. An association between LUTS and hypovitaminosis D, and between hypovitaminosis D and type 2 diabetes (T2DM), has also been suggested. Thus, we aim to evaluate possible relationships between hypovitaminosis D, LUTS, and BPH in T2DM men. In this prospective observational study, 67 T2DM males (57.9 ± 9.28 years) underwent medical history collection, International Prostate Symptom Score (IPSS) questionnaire, that allows the identification and grading of LUTS, physical examination, biochemical/hormonal blood tests (fasting plasma glucose, glycated haemoglobin, total cholesterol, high-density lipoprotein cholesterol, triglycerides, creatinine, LH, total testosterone, estradiol (E2 ), 25-OH-vitamin D, PTH, calcium, phosphate, and PSA) and ultrasound transrectal prostate examination. Subdividing patients into three groups, on the base of 25-OH-vitamin D concentration (sufficiency ≥50; insufficiency >25 < 50; and deficiency ≤25 nm), a significant progressive increase of prostate volume (p = 0.037), IPSS score (p = 0.019), diastolic blood pressure (p = 0.018), and a significant decrease in HDL cholesterol (p = 0.038) were observed. 25-OH-Vitamin D levels were inversely correlated with both IPSS (R = -0.333; p = 0.006) and prostate volume (R = -0.311; p = 0.011). At multivariate analysis, hypovitaminosis D remained an independent predictor of both IPSS and prostate volume. In conclusion, we showed, for the first time, an association between 25-OH-vitamin D deficiency, LUTS, and BPH in T2DM men.

HPV-DNA sperm infection and infertility: from a systematic literature review to a possible clinical management proposal.

The objectives of this study were to investigate the implications of human papillomavirus (HPV) sperm infection on male fertility, impairment of sperm parameters, and possible alteration of sperm nuclear status and to identify a possible effective management of infertile men with HPV sperm infection. We employed a systematic review and clinical management proposal at the Centers for Reproductive and Health care for treating infertile male patients with HPV infection. Literature search was carried out in electronic databases in the last two decades. We focused our attention on: (i) HPV sperm prevalence (ii) HPV-related alteration of sperm parameters; (iii) molecular mechanisms of HPV semen infection and infertility. The main outcome measures were HPV prevalence in infertile male patients and semen parameters. The prevalence of HPV sperm infection ranges between 2 and 31% in men from general population and between 10 and 35.7% in men affected by unexplained infertility. The presence of HPV in semen is associated with an impairment of sperm motility and the presence of anti-sperm antibodies. The molecular mechanisms underlying impairment of sperm motility apparatus need further evaluations. A greater attention should be applied to assess HPV sperm infection, particularly in men undergoing assisted reproduction techniques cycle for male infertility or sperm banking. It would be useful to perform HPV test and fluorescent in situ hybridization analysis for HPV in semen from these patients both at first admission, to define the possible presence and localization of semen infection, and after 6 months, to assess the possible virus clearance retrieval on normal sperm parameters.

Rare diseases in clinical endocrinology: a taxonomic classification system.

PURPOSE: Rare endocrine-metabolic diseases (REMD) represent an important area in the field of medicine and pharmacology. The rare diseases of interest to endocrinologists involve all fields of endocrinology, including rare diseases of the pituitary, thyroid and adrenal glands, paraganglia, ovary and testis, disorders of bone and mineral metabolism, energy and lipid metabolism, water metabolism, and syndromes with possible involvement of multiple endocrine glands, and neuroendocrine tumors. Taking advantage of the constitution of a study group on REMD within the Italian Society of Endocrinology, consisting of basic and clinical scientists, a document on the taxonomy of REMD has been produced. METHODS AND RESULTS: This document has been designed to include mainly REMD manifesting or persisting into adulthood. The taxonomy of REMD of the adult comprises a total of 166 main disorders, 338 including all variants and subtypes, described into 11 tables. CONCLUSIONS: This report provides a complete taxonomy to classify REMD of the adult. In the future, the creation of registries of rare endocrine diseases to collect data on cohorts of patients and the development of common and standardized diagnostic and therapeutic pathways for each rare endocrine disease is advisable. This will help planning and performing intervention studies in larger groups of patients to prove the efficacy, effectiveness, and safety of a specific treatment.

Prostate volume and growth during testosterone replacement therapy is related to visceral obesity in Klinefelter syndrome.

OBJECTIVE: Klinefelter syndrome (KS) is a chromosomal alteration characterized by increased risk of metabolic syndrome, mainly caused by visceral obesity. In the last years, obesity has been studied as a potential risk factor for prostate disease and recently a link has been demonstrated between visceral adiposity with prostate volume. The aim of this study was to analyze the relationship between obesity and prostate volume and growth during testosterone therapy in KS subjects. DESIGN AND METHODS: We evaluated reproductive hormones, metabolic parameters, anthropometric measures, PSA, and prostate volume in 121 naïve non-mosaic KS patients and 60 age-matched healthy male controls. Fifty-six KS hypogonadic subjects were treated with testosterone-gel 2% and reevaluated after 18 months of treatment. RESULTS: Prostate volume in KS was positively related to waist circumference (WC). The KS group with WC ≥94 cm had significantly higher prostate volume, BMI, insulin plasma levels, homeostasis model assessment index, total cholesterol, triglycerides, and glycemia with respect to the KS group with WC <94 cm. After testosterone replacement therapy, only hypogonadic KS men with WC ≥94 cm had a statistically significant increase in prostate volume. Furthermore, in untreated KS subjects, prostate volume showed a statistically significant increase after 18 months of follow-up only in subjects with WC ≥94 cm. CONCLUSIONS: This study showed that visceral obesity, insulin resistance, and lipid and glucose metabolism alterations are associated with prostate volume and growth during testosterone replacement therapy in KS, independently from androgen or estrogen levels. These latter findings might provide the basis for a better management and follow-up of KS subjects.

Human olfactory sensitivity for bourgeonal and male infertility: a preliminary investigation.

Olfactory receptor (OR) expression is also present in the sperm cells and could mediate sperm chemotaxis. OR1D2 was the first OR expressed in the testis demonstrated to be involved in chemotaxis and to be expressed also in the nose with a similar behavior. Bourgeonal is the OR1D2 most potent known agonist. Infertility affects ~15 % of couples in western countries and sometimes it is unexplained. This pilot study compared the bourgeonal olfactory thresholds, the ability of sperm to sense the bourgeonal and the frequency of 13 single nucleotide polymorphisms (SNPs) of OR1D2 gene in nine males suffering of unexplained infertility with a control group of 15 healthy males. The mean olfactory threshold for bourgeonal was statistically different between the study group (10.5 ± 3.7; median 12.3) and the control group (14.0 ± 2.8; median 15.5) (p = 0.006). Statistical analysis showed a significantly higher percentage of spermatozoa that migrated toward the capillaries filled with bourgeonal in the control group compared to the study group (p < 0.0001). Sperm migration was equally inhibited in both groups of subjects when, together with bourgeonal, capillaries were filled with undecanal, a strong bourgeonal inhibitor (p = 0.42). The 13 SNPs of OR1D2 revealed a statistically significant difference for allele and genotype frequency of rs769423 in study group versus control group (p = 0.02). The present preliminary study seems to confirm the important role of OR1D2 both in nose and spermatozoa and may explain the idiopathic infertility of the study group. Further studies on larger series are mandatory to confirm our preliminary evidence.

Altered bone status in unilateral testicular cancer survivors: Role of CYP2R1 and its luteinizing hormone-dependency.

BACKGROUND: Recent data suggest a potential role of testis in vitamin D activation, where Leydig cells could represent key players in this process since they express the highest amount of CYP2R1, a key enzyme involved in vitamin D 25 hydroxylation. AIM: To evaluate bone status in unilateral orchiectomy and to assess in vivo and in vitro LH-dependency of Vitamin D 25 hydroxylation. SUBJECTS AND METHODS: 125 normotestosteronemic patients with testicular cancer (TC), featured by unilateral orchiectomy and 41 age-matched healthy male controls were studied in the Center for Human Reproduction Pathology at the University of Padova. To evaluate LH-dependency of Vitamin D 25 hydroxylation in vitro, Leydig cell cultures were stimulated with hCG and assessed for CYP2R1 expression, whereas in vivo 10 hypogonadotropic hypogonadal (HH) patients were evaluated before and after treatment with gonadotropins for bone metabolism markers. Hormonal pattern and bone metabolism markers were measured in all subjects, whereas 105 patients and 41 controls underwent bone densitometry by DEXA. RESULTS: In TC patients 25-hydroxyvitamin D levels were significantly lower compared to controls. Furthermore, 23.8% of patients with TC displayed low bone density (Z-score <-2 SD). None of the 41 control subjects showed any significant alteration of BMD. In vitro and in vivo studies revealed that CYP2R1 expression in Leydig cells appeared to be hCG dependent. CONCLUSION: Our data show an association between TC and alteration of the bone status, despite unvaried androgen and estrogen levels, suggesting the evaluation of bone status and possible vitamin D deficiency in TC survivors.

Platelets express and release osteocalcin and co-localize in human calcified atherosclerotic plaques.

BACKGROUND: Although vascular-calcification mechanisms are only partially understood, the role of circulating calcifying cells and non-collagenous bone matrix proteins in the bone-vascular axis is emerging. In spite of the fact that platelets represent a cellular interface between hemostasis, inflammation and atherosclerosis, and have a myeloid precursor, a possible involvement in the modulation of vascular calcification has rarely been investigated. We investigated if osteocalcin (OC) is released by platelets and described OC expression in patients with carotid artery occlusive disease. METHODS: Expression and release of OC were determined by Western blot, immunofluorescence, fluorescence-activated cell sorting (FACS) and ELISA in human resting and activated platelets and megakaryocytes. Co-localization of platelet aggregates, macrophages, OC and calcifications was studied in carotid endarterectomy specimens and normal tissues. RESULTS: Human platelets expressed OC and co-localized with CD63 in δ-granules. Upon activation with an endogenous mechanism, platelets released OC in the extracellular medium. Expression of OC in megakaryocytes suggested lineage specificity. The OC count in circulating platelets and the released amount were significantly higher in patients with carotid artery occlusive disease than in healthy controls (P < 0.0001) in spite of similar serum levels. In atherosclerotic plaques, OC strongly overlapped with CD41+ platelets in the early stage of calcification, but this was not seen in normal tissues. CD68+OC+ cells were present at the periphery of the calcified zone. CONCLUSIONS: Given the active role played by platelets in the atherosclerotic process, the involvement of OC release from platelets in atherosclerotic lesions and the impact of genetic and cardiovascular risk factors in mediating bone-marrow preconditioning should be investigated further.

Effects of endogenous FSH on normal human spermatogenesis in adults.

Unilateral orchiectomy (UO) in adult bonnet monkeys and boars elicits a compensatory increase in size and sperm production of the remaining testis. The objective of this study was to investigate whether a similar effect is evident also in humans. We prospectively studied 50 patients from October 2003 to December 2005 who underwent UO for seminomatous tumour, with sperm concentration >20 × 10(6) /mL or total sperm count >40 × 10(6) at diagnosis and without elevation of serum tumour markers. Patients were followed-up with surveillance and they were studied at the time of diagnosis of testicular cancer (T(-1) ), 1 month after unilateral orchiectomy (T(0) ) and yearly for 3 years (T(1) , T(2) , T(3) ) with semen analysis, measurement of plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), inhibin B, total testosterone, and oestradiol and ultrasonographic scanning of the remaining testis. A decline in circulating inhibin B and an increase in FSH levels were evident 1 month after UO. The elevation of FSH was maintained up to 3 years and was associated with a significant increase in testicular volume of 19 and 30%, 2 and 3 years after UO respectively. Although patients had normozoospermia at the time of diagnosis of testicular cancer, they showed a statistically significant increase in total sperm count at T(2) and T(3) with respect to T(-1) and T(0.) In conclusion, we showed that in humans, the testes are not normally operating at their maximal potential in terms of spermatogenesis. Therefore, in physiological situations, FSH secretion is insufficient to stimulate spermatogenesis to its ceiling. A sustained endogenous increase in FSH secretion might drive human testes towards their maximal function.

Human papilloma virus in the sperm cryobank: an emerging problem?

Human papilloma virus (HPV) infection is very common worldwide, but the actual incidence and significance of HPV infection in sperm are poorly understood. In this study, we evaluated the presence of HPV in spermatozoa from thawed semen samples previously stored in our sperm bank. We performed polymerase chain reaction and in situ hybridization for HPV detection in cryovials belonging to 98 oncology patients and in 60 semen samples from healthy controls. Statistical analysis was performed by two-tailed Student's t-test and Fisher's exact test. The frequency of HPV semen infection was 6.1% in thawed cryovials from patients and 3.3% in semen samples from controls. Among the patients, four were found positive for high-risk HPV, one for medium-risk HPV and another for low-risk HPV. Patients had a significantly higher percentage of infected sperm than controls. In conclusion, this report shows the presence of HPV in sperm cells from cryovials of a sperm bank. It is still unclear if HPV-infected sperm are able to cross-contaminate cryovials and impair the outcome of assisted reproduction techniques or to infect partners. Further studies are needed to understand whether screening for HPV should be performed in all semen samples before sperm banking or before intra-cytoplasmic sperm injection procedures.

Increased osteocalcin-positive endothelial progenitor cells in hypogonadal male patients.

OBJECTIVE: Endothelial dysfunction is considered a key factor in the development of cardiovascular diseases. Endothelial regeneration is necessary for the maintenance of endothelial function and circulating endothelial progenitor cells (EPC) participate of it in both direct and indirect manner. The molecular phenotype of EPC is not univocally defined and recent studies identified an osteocalcin (OCN)-positive (EPC-OCN+) subpopulation of EPC highly correlated with atherosclerosis progression. AIM: Considering that hypogonadism is a risk factor for cardiovascular diseases and atherosclerosis, we investigated the circulating levels of EPC-OCN+ in hypogonadal patients. SUBJECTS AND METHODS: Ten hypogonadotropic hypogonadal (HH) male patients and 30 healthy eugonadal men were evaluated for clinical status and hormonal levels. Circulating levels of CD34+/CD133+/kinase insert domain-receptor+ EPC and EPC-OCN+ were also determined by flow cytometry. RESULTS: Compared to controls, HH patients displayed lower FSH, LH, estradiol, testosterone, and EPC levels. On the contrary, EPC-OCN+ were significantly increased. CONCLUSIONS: The observed association of low levels of circulating EPC and increased values of EPC-OCN+ sub-population in hypogonadal men strengthens the significance of hypogonadism as cardiovascular risk factor.

Clinical use of testicular fine needle aspiration cytology in oligozoospermic and azoospermic dogs.

Clinical investigation of canine testicular function is complicated by the difficulty in the evaluation of seminiferous tubules. Until recently, testicular biopsy was the only diagnostic option for dogs with persistent oligo/azoospermia. In human andrology, testicular fine needle aspiration (TFNA) is currently considered a useful method in the evaluation of azoospermia and severe oligozoospermia, and has long replaced classical biopsy to evaluate spermatogenesis. In order to verify its diagnostic efficacy for the clinical approach to canine oligo- or azoospermia, TFNA was performed in seven adult (two oligozoospermic and five azoospermic) dogs. After sedation, a fine (21-23 gauge) butterfly needle connected to a 50-ml syringe was inserted into each testicle; strong suction was applied and the aspirated fluid squirted on a glass slide, smeared out, air-dried and stained with a modified May-Grunwald-Giemsa. Under light microscopy, Sertoli cells (all those found in each investigated field) and spermatogenic cells (n = 100) were counted on each smear in order to differentiate spermatogonia, primary spermatocytes, secondary spermatocytes, early spermatids, late spermatids and spermatozoa, and calculate their relative percentages. Cytological analysis showed the following testicular pictures: normal spermatogenesis (compatible with obstruction of the seminal ducts), hypospermatogenesis, maturative disturbances and Sertoli cell-only syndrome. Two dogs with an obstructive lesion were treated with corticosteroids; one of them recovered and sired two litters of puppies.

Mutations in dynein genes in patients affected by isolated non-syndromic asthenozoospermia.

BACKGROUND: Asthenozoospermia (AZS) is a common cause of male infertility characterized by reduced forward motility (WHO grade A+B sperm motility <50% or A < 25%) or absent sperm motility in fresh ejaculate. AZS may exist as an isolated disorder, in combination with other sperm anomalies or as part of a syndromic association. Up to date, only a few genes, constituting the cilia/flagella structure, have been associated with isolated AZS in humans, whereas several other genes are known to be involved in syndromic form of AZS, including primary ciliary dyskinesia (PCD) and Kartagener syndrome (KS). Axonemal ultrastructural defects, including absent or shortened arms of dyneins, can be found in >50% of PCD/KS patients. Approximately 90% of KS male patients are affected by AZS. The majority of KS patients can be ascribed to dynein genes mutations. METHODS: Mutation screening of DNAI1, DNAH5 and DNAH11 genes was performed in 90 patients with isolated non-syndromic AZS and 200 controls. RESULTS: We found three mutations (one in each gene) specifically associated with AZS in seven patients (7.8%). Mutations are inherited from the mothers and may be found in familial clusters. No ultrastructural axonemal anomaly was detected in sperm. CONCLUSIONS: We report for the first time a possible association between mutations in dynein genes and isolated AZS. Male carriers of the mutations always exhibit AZS, whereas female carriers manifest no alterations in either fertility or pulmonary clearance.

Y chromosome haplogroups and susceptibility to testicular cancer.

Although in the past decades much progress in testicular cancer (TC) management has been made, little is known about the possible genetic causes and molecular mechanisms involved in its aetiopathogenesis. Some studies on possible contribution of the Y chromosome in TC development have been previously published, but data are not conclusive. In particular, ethnic influence and spermatogenic activity of patients with TC have not been adequately considered in previous studies, although they may represent important confounding factors. The objective of this study is to analyse the contribution of the Y chromosome in testicular germ cell cancer subjects who are well defined at the microgeographical, clinical and seminological level. We analysed Y chromosome classic azoospermia factor (AZF) deletions, partial AZFc deletions and Y haplogroups in 118 sporadic cases of testicular germ cell cancer and 93 microgeographically matched controls. Y chromosome screening failed to identify Y chromosome microdeletions in either cases or controls. Y chromosome haplogroup distribution and frequencies did not differ between cases and controls. Furthermore, no difference was observed when comparing patients with seminoma and non-seminoma, nor when comparing patients with TC with normozoospermia and azoo-oligozoospermia. Our findings combined with data reported so far suggest that classic AZF deletions and partial AZFc deletions are not a frequent cause or risk factor for TC and that different Y haplogroup distribution does not contribute to susceptibility to this tumour.